Fig. 4.

Flow scheme of the cytokine OL protection assay. A primary O4-positive OPCs were isolated for P6–P7 neonatal rat brains. A OPCs were expanded in flasks for 5–7 days in defined media supplemented with PDGF. B Expanded OPCs were plated in 96-well plates in defined media supplemented with T3, but without PDGF. C Test compounds were added following a 24-h incubation period followed 1 h later by addition of cytokine challenge (1 ng/ml TNFα+10 U/ml IFNγ). D After 48-h culture, alamarBlue® (AB) is added and AB fluorescence used to determine viability and metabolic activity of the cells. After selection of hit compounds (>50 % of the positive control, 1.1 µM QTP), fresh compound from a new source was obtained and the dose response relationship and EC₅₀ values of each compound were determined. Following AB read out of the dose response, cells were fixed, immunostained, and images quantified to determine OL differentiation (MBP expression)